The show's theme song features the voices of all six leading characters, and is played in every episode's title sequence. Shortened instrumental versions of the theme song play on the menus of the show's DVDs, and modified versions of the theme song play in some of the brand's toy commercials. The song is also included in the My Little Pony Live Show.

The opening lyrics and melody are borrowed from the theme of the original My Little Pony television series, which ran from 1986 to 1987.

Except Italian season one[note 1] and the Japanese version which both have their own themes, most of the non-English versions, including Boomerang and Cartoonito Italy's broadcasts of subsequent seasons, use translations of this song.

A remastered version of the theme song debuted in Lesson Zero.[2] It features unaltered lyrics, but Rebecca Shoichet's vocals for Twilight Sparkle are re-recorded, the instruments are higher in the mix in relation to the vocals, it features a rising "whoop" sound as Rainbow Dash spins through the clouds, and it is accompanied by a slightly different animated sequence. "Creme Brulee" is replaced by Big McIntosh, Granny Smith walks in the opposite direction, there is an addition of a train station and a steam-engine Friendship Express train running through Ponyville, with Derpy peeking from one of the cars' windows and the Cutie Mark Crusaders sitting in the last car, and Princess Celestia's magic now has its proper yellow glow. The melody of the first theme can still be heard in the end credits for most episodes.

A slightly updated version is used starting from Sisterhooves Social, which rearranges some of the Friendship Express train cars, changes one of them from brown to purple, and changes the locomotive and caboose from blue to pink.

The theme song has a roughly two-minute extended set of lyrics. A small portion of the extended version of the song was originally introduced as one of three songs included with the My Little Pony Twilight Sparkle Animated Storyteller, an animatronic, plush toy in the form of Twilight Sparkle.[5] An instrumental version of an earlier portion is played in My Little Pony Friendship is Magic: Discover the Difference.

The extended version was eventually released in sing-a-long form via Shout! Factory's My Little Pony Friendship is Magic: The Friendship Express[6] and My Little Pony Friendship is Magic: Season One Disc 4 Region One DVDs, as parts of the extra content, and was also released in sizzle reel form. The sing-a-long video was also included in Clear Vision's My Little Pony Friendship is Magic: Welcome to Ponyville Region Two DVD.

The opening rhythm in the song echoes the classical My Little Pony theme. The original 10-note theme that is heard in all "My Little Pony" themes was written by Anna Bryant, Clifford "Ford" Kinder, and Barry Harman.[7] Lyrics sung by the other main characters are not included in the extended version.

A slightly shortened version of this serves as the third closing theme for the Japanese broadcast of Friendship is Magic.